Male ddY mice weighing 20–25 g were used throughout the experiments. The mice were housed in a room maintained at 21 ± 2°C, 55 ± 5 % relative humidity and an automatic 12-h light/dark cycle with free access to standard laboratory diet and tap water. The animals were adapted to the testing environment (maintained at 21 ± 2°C, 55 ± 5 % relative humidity and 12-h light/dark cycle) by keeping them in the testing room 24 h before the experiments. Experiments were performed during the light phase of the cycle (10:00 – 17:00). All procedures were approved by Nagasaki University Animal Care Committee and complied with the recommendations of International Association for the Study of Pain .
Drugs and injection methods
Following drugs were purchased: morphine hydrochloride (Takeda Pharma. Co. Ltd., Japan), naloxone hydrochloride, methysergide maleate, phentolamine hydrochloride, fluoxetine hydrochloride, and clonidine hydrochloride (all from Sigma Co., St Louis, MO, USA). All drugs were dissolved in physiological saline. Physiological saline was used for control injections. The intrathecal (i.t.) injections were performed free hand between spinal L5 and L6 segments according to the method of Hylden and Wilcox . The intracerebroventricular (i.c.v.) injections were carried out into the left lateral ventricle of mice. Injections were performed using a Hamilton microsyringe fitted with a 26-gauge i.c.v. needle, according to the method of Haley and McCormick . The site of injection was 2 mm caudal and 2 mm lateral to the bregma, and 3 mm in depth from the skull surface. Both i.t. and i.c.v. injections were given in a volume of 5 μl. The mice received the subcutaneous (s.c.) injections in a volume of 0.1 ml/10 g body weight.
Partial ligation of sciatic nerve
Partial ligation of the sciatic nerve of mice was performed under pentobarbital (50 mg/kg i.p.) anesthesia, following the methods of Malmberg and Basbaum . Briefly, the common sciatic nerve of the right hind limb of mice was exposed at high thigh level through a small incision and dorsal 1/3 to 1/2 of the nerve thickness was tightly ligated with a silk suture. The wound was closed with a single muscle suture and antibiotic powder was dusted over the wound area following surgery. Sham operation was performed similarly except without touching the sciatic nerve. Immediately following surgery, the animals were kept in a soft bed cage with some food inside so that the animals could feed themselves without difficulty in standing. The wound healed within 1–2 days and the animals behaved normally. Experiments were carried out at 7 or 14 days post-ligation.
Hargreaves thermal paw withdrawal test
Analgesia was measured from the latency to withdrawal evoked by exposing the right hind paw to a thermal stimulus. Mice were placed under Plexiglas cages on top of a glass sheet. The thermal stimulus (IITC Inc., Woodland Hills, CA, USA) was positioned under the glass sheet to focus the projection bulb exactly on the middle of plantar surface of the animals. A mirror attached to the stimulus permitted visualization of the undersurface of the paw. After one hour of adaptation, paw withdrawal latencies were measured at every 10 min interval until 60 min with vehicle or drug pretreatment. A cut-off thermal latency of 20 s was set in order to prevent tissue damage.
Paw pressure test
Experiments were performed as described previously . Briefly, mice were placed under a Plexiglas chamber on a 6 mm × 6 mm wire mesh grid floor and were allowed to accommodate for a period of one hour. The mechanical stimulus was then delivered onto the middle of the plantar surface of right hind-paw using a Transducer Indicator (Model 1601, IITC Inc., Woodland Hills, USA). The paw withdrawal thresholds were measured at every 10 min interval until 60 min with vehicle or drug pretreatment. In this experiment, a cut-off pressure of 20 g was set to avoid tissue damage.
DAB immunostaining for c-Fos
Mice were deeply anesthetized with i.p. pentobarbital and perfused transcardially with 40 ml of 0.1 M potassium free phosphate buffered saline (K+ free PBS, pH 7.4) followed by 40 ml of 4% paraformaldehyde (PFA) in 0.1 M K+ free PBS. The spinal cord between L4 – L5 segments was removed and post-fixed in 4% PFA for 1 hour. Then, the sample was transferred to 25% sucrose solution (in 0.1 M K+ free PBS) overnight for cryoprotection. Next day, the spinal cord sample was fast-frozen in cryoembedding compound on a mixture of ethanol and dry-ice and stored at -80°C until use. The spinal cord sample was cut as 20 μm thick transverse sections with a cryostat, thaw-mounted on silane-coated glass slide and air dried overnight at room temperature (RT). For c-Fos immunolabeling, spinal cord sections were washed 3 times with K+ free PBS for 5 min each then incubated in excess 100% methanol with 0.1% H2O2. After 3 washings with K+ free PBS, the sections were incubated in excess blocking buffer containing 10% normal goat serum and 2% bovine serum albumin in PBST (2% NaCl, 0.1% Triton-X 100 in K+ free PBS) for 60 min at RT. The sections were washed and reacted with rabbit polyclonal antibody raised against the c-Fos protein (1:1000 in 2% BSA in PBST solution; sc-7202, Santa Cruz Biotechnology, CA, USA) at 4°C overnight. After thorough washings, the sections were incubated with secondary antibody, biotinylated anti-rabbit IgG (1:200 in 2% BSA in PBST solution; Vector, CA, USA) at RT for 60 min, and subsequently with ABC complex (Vector, CA) at RT for 60 min. The antigen-antibody reaction sites were visualized by incubation with a solution containing 0.005% 3,3'-diaminobenzidine tetrahydrochloride (DAB; Dojindo, Japan), 0.002% H2O2, 0.001% nickel ammonium sulfate and 0.002% cobalt chloride in 0.1 M K+ free PBS until the black reaction products appear. The reaction was stopped by washing with ice-cold PBS. After 3–4 washings, the sections were cover-slipped and visualized under a light microscope. The number of c-Fos-positive cells in the ipsi-and contralateral sides of dorsal horn gray mater of the spinal cord was then counted from lamina I-VI and plotted in a bar graph.
Fluorescence immunohistochemistry for PKCγ
The spinal cord sections were prepared as described above. For immunostaining of PKCγ, the spinal cord sections were first pre-blocked with blocking buffer containing 10% normal goat serum and 2% bovine serum albumin in PBST. The sections were then reacted with a rabbit polyclonal antibody raised against the γ isoform of protein kinase C (1:500 in 2% BSA in PBST solution; sc-211, Santa Cruz Biotechnology, CA, USA) at 4°C overnight. The sections were then incubated with a FITC-conjugated anti-rabbit IgG (1:200; Santa Cruz Biotechnology) for 60 min at RT. The sections were washed thoroughly, cover-slipped with Perma Fluor (Thermo Shandon, Pittsburgh, PA, USA) and examined under a fluorescence microscope (Olympus, Tokyo, Japan). Quantification of the intensity of PKCγ-positive fluorescence was then done using Scion imaging software for Macintosh (Scion Corporation, USA).
Statistical evaluations of the data were performed by comparison with repeated measures analysis of variance (ANOVA) with suitable post-hoc tests. The criterion of significance was set at p < 0.05. All results are expressed as the mean ± SEM.